102 3.6  Basic Fluorescence Microscopy Illumination Modes

Also, a range of fluorophores are now available whose fluorescence lifetime is dependent

on the local viscosity of their cellular environment (Kuimova, 2008). These dye molecules typ­

ically operate by undergoing periodic mechanical transitions as nanoscale rotors in forming a

transient electrical dipole that can absorb excitation light. As with all dyes, each molecule will

emit a characteristic approximate number of photons prior to irreversible photobleaching

most likely due to free radical chemical damage of the dye. Since the frequency of rotation of

the dye is a function of local viscosity, the dye fluorescence lifetime is therefore a metric for

viscosity, and thus FLIM measurements using such dyes can map out viscosity over single

cells. This is important since local cellular viscosity is a manifestation of the underlying

subcellular architecture in that specific region of the cell and thus gives us insight into these

different biological features at the nanoscale.

There are also several fluorophores available whose fluorescence emission output is par­

ticularly sensitive to specific chemical and physical environmental conditions. Into this

category can be included voltage-​sensitive dyes and probes which can measure molecular

crowding (for example, a FRET pair of fluorescent protein molecules attached by a lever arm

which closes to give high FRET efficiency at high molecular crowding conditions but opens

out to give a lower FRET efficiency at low molecular crowding. But other dyes also exist,

which have been chemically optimized to be highly sensitive to local pH or the binding of

ions such calcium (Ca^2+​), whose fluorescence intensity and fluorescence lifetime change in

response to binding. These dyes therefore act as nanoscale environmental sensors, and FILM

can map out the absolute values of these environmental parameters in the cell. Many of these

dyes operate through having specific regions of their emission spectra, which are sensitive

to environmental change, whereas other regions of the emission spectrum may be relatively

insensitive. Usually, therefore, a ratiometric approach is taken to measure the relative ratio of

emission intensity change at the sensitive and insensitive regions of the emission spectrum,

since this ratio will no longer be sensitive to absolute concentrations of the dye in a given

localization of the cell.

Direct measurement of the integrated fluorescence intensity of individual dye molecules

can also be used as a metric for the physical and chemical environment, that is, the total

brightness of a dye molecule is a function of several different environment factors, depending

upon the specifics of the dye. A more precise metric is to perform spectral imaging of the

dye molecule. Here, the fluorescence emission signal can be directed through a transmission

diffraction grating, such that the zeroth order (undeviated light) can be imaged onto one-​half

of a camera detector, while the first order (deviated light) is imaged onto the other half. The

zeroth order can be used to determine precisely where the molecule is by using localization

fitting algorithms (discussed in Chapter 4) while the first order is a measurement of the trans­

mission spectrum of that dye molecule, since the diffraction angle is wavelength dependent.

Thus, the 1D profile of this spectral image can therefore be used as a very precise indicator

for local environmental parameters.

KEY POINT 3.4

Fluorescent dyes are often sensitive to many local physical and chemical parameters.

Such dyes can be optimized so that they can be used as direct reporters for the output

of these physicochemical parameters in live cells, observing changes to fluorescence

lifetimes, through a direct or ratiometric intensity approach of fluorescence emissions

or through direct spectral imaging.

3.6.6  SLIMFIELD AND NARROW-​FIELD EPIFLUORESCENCE MICROSCOPY

Biological molecules typically diffuse much faster inside the watery innards of cells than in

membrane surfaces due to lower effective viscosities in the cell cytoplasm by two to three

orders of magnitude, and they have a high likelihood of moving during each sampling time